Differentiation of whole bone marrow

ABSTRACT

A method is described for generating a clinically significant volume of neural progenitor cells from whole bone marrow. A mass of bone marrow cells may be grown in a culture supplemented with fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF). Further methods of the present invention are directed to utilizing the neural progenitor cells cultured in this fashion in the treatment of various neuropathological conditions, and in targeting delivery of cells transfected with a particular gene to diseased or damaged tissue.

This application claims the benefit of priority under 35 U.S.C. § 119(e)of provisional application Ser. No. 60/334,957, filed Oct. 25, 2001, thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

Embodiments of the present invention are directed to a method forgenerating a clinically substantial volume of neural progenitor cellsfrom mammalian whole bone marrow. Further embodiments of the presentinvention are directed to the treatment of neurological disorders usingneural progenitor cells cultured in this fashion.

BACKGROUND OF THE INVENTION

Nearly every cell in an animal's body, from neural to blood to bone,owes its existence to a stem cell. A stem cell is commonly defined as acell that (i) is capable of renewing itself; and (ii) can give rise tomore than one type of cell (that is, a differentiated cell) throughasymmetric cell division. F. M. Watt and B. L. M. Hogan, “Out of Eden:Stem Cells and Their Niches,” Science, 284, 1427–1430 (2000). Stem cellsgive rise to a type of stem cell called progenitor cells; progenitorcells, in turn, proliferate into the differentiated cells that populatethe body.

The prior art describes the development, from stem cell todifferentiated cells, of various tissues throughout the body. U.S. Pat.No. 5,811,301, for example, the disclosure of which is herebyincorporated by reference, describes the process of hematopoiesis, thedevelopment of the various cells that comprise blood. The process beginswith what may be a pluripotent stem cell, a cell that can give rise toevery cell of an organism (there is only one cell that exhibits greaterdevelopmental plasticity than a pluripotent stem cell; this is afertilized ovum, a single, totipotent stem cell that can give rise to anentire organism when implanted into the uterus). The pluripotent stemcell gives rise to a myeloid stem cell. Certain maturation-promotingpolypeptides cause the myeloid stem cell to differentiate into precursorcells, which in turn differentiate into various progenitor cells. It isthe progenitor cells that proliferate into the various lymphocytes,neutrophils, macrophages, and other cells that comprise blood tissue ofthe body.

This description of hematopoiesis is vastly incomplete, of course:biology has yet to determine a complete lineage for all the cells of theblood (e.g., it is has yet to identify all the precursor cells betweenthe myeloid stem cell and the progenitor cells to which it gives rise),and it has yet to determine precisely how or why the myeloid celldifferentiates into progenitor cells. Even so, hematopoiesis isparticularly well studied; even less is known of the development ofother organ systems. With respect to the brain and its development, forexample, U.S. Pat. No. 6,040,180, the disclosure of which is herebyincorporated by reference, describes the “current lack of understandingof histogenesis during brain development.” U.S. Pat. No. 5,849,553, thedisclosure of which is hereby also incorporated by reference, describesthe “uncertainty in the art concerning the development potential ofneural crest cells.”

The identification and isolation of stem cells has daunted researchersfor decades. To date, no one has identified an individual neural stemcell or hematopoietic stem cell. F. H. Gage, “Mammalian Neural StemCells,” Science, 287, 1433–1488 (2000). There are two principaldifficulties. First, stem cells are rare. In bone marrow, for example,where hematopoiesis occurs, there is only one stem cell for everyseveral billion bone marrow cells. G. Vogel, “Can Old Cells Learn NewTricks?” Science, 287, 1418–1419 (2000). Second, and more importantly,researchers have been unable to identify molecular markers which areunique to stem cells; to the typical immunoassay, most stem cells looklike any other cell. Id. Compounding this problem is that primitive stemcells may be in a quiescent state. As a result, they may express fewmolecular markers. F. H. Gage, supra.

A method to effectively isolate stem cells and culture them inclinically significant quantities would be of immense importance.Researchers are already transplanting immature neurons, presumed tocontain neural stem cells, from human fetuses to adult patients withneurodegenerative disease. The procedure has reduced symptoms by up to50% in patients with Parkinson's disease in one study. M. Barinaga,“Fetal Neuron Grafts Pave the Way for Stem Cell Therapies,” Science,287, 1421–1422 (2000). Many of the shortcomings of this procedure,including the ethical and practical difficulties of using materialderived from fetuses and the inherent complications of harvestingmaterial from adult brain tissue, could be addressed by using culturesof isolated stem cells, or stem cells obtained from adult individuals.D. W. Pincus et al., Ann. Neurol. 43:576–585 (1998); C. B. Johansson etal., Exp. Cell. Res. 253:733–736 (1999); and S. F. Pagano et al., StemCells 18:295–300 (2000). However, the efficient and large-scalegeneration of neural progenitor cells for use in the treatment ofneurological disorders has been a challenge.

Recent evidence has suggested that progenitor cells outside the centralnervous system and bone marrow cells in paricular may have the abilityto generate either neurons or glia in vivo. J. G. Toma et al., Nat. CellBiol. 3:778–783 (2001); E. Mezey et al., Science 290:1779–1782 (2000);T. R. Brazleton et al., Science 290:1775–1779 (2000); and M. A. Eglitiset al., Proc Natl. Acad. Sci. 94:4080–4085 (1997). Bone marrow stromalcells have also been shown to be capable of differentiating into neuronsand glia in vitro after a complicated and time-consuming culture processspanning several weeks. The generation of neural progenitor cells fromwhole bone marrow has, however, not been reported.

SUMMARY OF THE INVENTION

The invention described herein provides an efficient method ofgenerating a clinically significant quantity of neural progenitor cells.These neural progenitor cells may be generated from bone marrow or otherappropriate sources, and may be used to treat a variety of conditions,particularly neuropathological conditions. Owing to the neuralprogenitor cells' ability to track diseased or damaged neural tissue andto further replace the lost function of such tissue, the cells of thepresent invention are particularly useful in the treatment of conditionswherein neural tissue itself is damaged.

Still further embodiments of the present invention describe the use ofthe neural progenitor cells to target the delivery of various compoundsto damaged or diseased neural tissue. Neural progenitor cells may becaused to carry a gene that induces the cells themselves to secrete suchcompounds, or to otherwise effect the local production of such compoundsby, for example, initiating or promoting a particular biochemicalpathway. Since the neural progenitor cells that carry these genes maytrack diseased or damaged neural tissue, delivery of the particularcompound may be correspondingly targeted to such tissue. A dualtreatment effect is accomplished when the neural progenitor cells bothreplace lost or damaged neural tissue function while simultaneouslyeffecting the targeted delivery of a therapeutic compound.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 depicts neural progenitor cells obtained from human bone marrowin accordance with an embodiment of the present invention. FIG. 1Adepicts cells from whole bone marrow that, when plated on poly-D-lysine,form a monolayer that gives rise to distinct cellular spheres after fourdays in culture. FIG. 1B depicts the spheres of FIG. 1A at highermagnification; cells may be easily collected, sub-cultured, andpropagated separately in the presence of growth factors. FIG. 1C depictsthat the spheres, once differentiated, attach and cells start migratingoutward (arrows indicate migrating cells). FIG. 1D depicts that theformed spheres detach from the bottom and afterwards remainfree-floating.

FIG. 2 is executed in color and depicts neural progenitor cells obtainedfrom human bone marrow in accordance with an embodiment of the presentinvention. FIGS. 2A and 2B indicate that neurospheres (i.e., spheresderived from neural cells) and bone marrow-derived spheres,respectively, were morphologically indistinguishable. FIGS. 2C and 2Dindicate that the pattern of nestin expression (red) was similar both inneurospheres and bone marrow derived spheres, respectively. Nuclei ofcells appear blue owing to being counterstained with4′,6-diamidino-2-phenylindole (DAPI).

FIG. 3 is executed in color and depicts neural progenitor cells obtainedfrom human bone marrow in accordance with an embodiment of the presentinvention. FIG. 3A indicates that the bone marrow-derived spheresexpressed the ectodermal marker vimentin. As depicted in FIG. 3B, a weakstaining for fibronectin was also observed in the neural progenitorcells. As depicted in FIG. 3C, bone marrow-derived spheres exhibitstrong expression of CD90, and, as depicted in FIG. 3D, the majority ofthe cells in spheres exhibit nuclear expression of Neurogenin 1.

FIG. 4 is executed in color and depicts a differentiation of bone marrowderived cells into neurons and glia in accordance with an embodiment ofthe present invention. After plating on a substrate in media devoid ofgrowth factors, the bone marrow-derived spheres attached, migrated awayfrom the primary site of attachment, and displayed multiplemorphologies, as depicted in FIG. 4A. FIGS. 4B and 4C depict neuralprogenitor cells of the present invention expressing the glial cellmarker glial fibrillary acidic protein (GFAP) after eight and nine daysof differentiation, respectively (cellular nuclei counterstained withDAPI). FIGS. 4D and 4E depict neural progenitor cells of the presentinvention expressing the neuronal marker Neuron Specific Enolase (NSE)after eight days of differentiation (cellular nuclei counterstained withDAPI). Scattered cells also expressed the later neuronal marker MAP2, asdepicted in FIG. 4F. After transplantation of the bone marrow derivedspheres into the hippocampus of a syngeneic animal, cells expressingNeuN were found, as depicted in FIG. 4G. Some of these cells appeared tointegrate into the hippocampal structure, as depicted in FIG. 4H. FIGS.4I, 4J and 4K depict a similar differentiation of bone marrow derivedcells, with alternate antibodies used for immunocytochemistry. FIG. 4Idepicts the use of the oligodendrocyte marker CNPase (1:400 Sigma) at40× magnification, while FIGS. 4J and 4K depict the use of the neuronalmarker NF200 (1:100 Chemicon) at 20× and 40× magnification,respectively.

FIG. 5 is executed in color and depicts a gene transfer to neuralprogenitor cells using a β-galactosidase gene-bearingreplication-deficient adenoviral vector in accordance with an embodimentof the present invention.

FIG. 6 is executed in color and depicts neural progenitor cells infectedwith green fluorescent protein (GFP) bearing double herpes simplex virustype I in accordance with an embodiment of the present invention.

FIG. 7 is executed in color and depicts neurospheres generated fromprimary fetal brain culture in accordance with an embodiment of thepresent invention. FIG. 7A depicts neural progenitor cells grown intospherical aggregates. FIG. 7B depicts nestin expression by theseneurospheres (nuclei counterstained with DAPI). Neurons expressed β-IIItubulin, astrocytes expressed GFAP, and oligodendrocytes expressedCNPase (FIGS. 7C, 7D, and 7E, respectively). FIG. 7F depicts expressionof β-galactosidase by neural progenitor cells infected in vitro withAdLacZ. Magnification 400× for FIGS. 7B, 7C, 7D, and 7E; 100× for FIGS.7A and 7F.

FIG. 8 is executed in color and depicts an intra-arterial delivery ofneural progenitor cells into an experimentally induced ischemic lesionin accordance with an embodiment of the present invention. Single cellsare distributed widely throughout the brain tissue (FIG. 8A).Transplanted cells exhibit tropism for injured basal ganglia (FIG. 8B;at 400× magnification).

FIG. 9 is executed in color and depicts neural progenitor cells trackingtumor cells in vivo in accordance with an embodiment of the presentinvention. FIG. 9A depicts a thin outgrowth of tumor cells deep intoadjacent normal brain. FIG. 9B depicts a direct extension of tumor massinto adjacent tissue. FIG. 9C depicts a migration of glioma cells awayfrom the primary tumor bed along a white matter tract. FIG. 9D depicts atumor microsatellite independent of a main tumor mass. FIG. 9E depicts ahigh power photomicrograph of the microsatellite depicted in FIG. 9D;further depicting β-galactosidase-positive neural progenitor cellsinterspersed with tumor cells. FIG. 9F shows an inoculation of neuralprogenitor cells (left panel) and a tumor mass (right panel) into whichneural progenitor cells migrated from the opposite hemisphere (insetbox). Neural progenitor cells appear blue (expressing β-galactosidase),whereas tumor cells appear red (hypercellular areas stained intensivelywith neural red). “T” represents tumor mass, outgrowths, andmicrosatellites. Arrows indicate disseminating neural progenitor cellsclosely following migrating pockets of tumor.

FIG. 10 is executed in color and depicts intratumoral CD4+ and CD8+T-cell infiltration in accordance with an embodiment of the presentinvention. FIG. 10A depicts a flow cytometry analysis demonstratingintratumoral T-cell infiltration in brain tissue treated with neuralprogenitor cells secreting IL-12 (left panel) and 3T3-IL-12 (centerpanel), and a comparative lack of infiltration in tissue treated withneural progenitor cells secreting LacZ (right panel). CD4+ (left panel)and CD8+ (right panel) intratumoral infiltration is depicted in tissuetreated with neural progenitor cells secreting 3T3-IL-12, LacZ, andIL-12 (FIGS. 10B, 10C, and 10D, respectively). Aggregates appeared alongthe tumor/normal tissue boundary in tissue treated with neuralprogenitor cells secreting IL-12 (FIG. 10D, arrows indicate aggregates).FIG. 10E depicts a comparison of T-cell infiltration in comparableoutgrowths from a primary tumor bed for tissue treated with neuralprogenitor cells secreting IL-12 and 3Y3-IL-12 (FIGS. 10E, left andright panels, respectively). “T” designates tumor and “N” designatesnormal brain tissue. Magnification 100× for FIGS. 10B, 10C, and 10D, and200× for FIG. 10E.

FIG. 11 is executed in color and depicts transplantation of neuralprogenitor cells expressing GFP into rat hippocampus in accordance withan embodiment of the present invention. FIG. 11A depicts a migration oftransplanted cells (green). FIG. 11B depicts individual cells expressingNSE (red) and GFP together with NSE (yellow). Transplanted cells werestained for NSE and exhibit GFP (green), NSE (red), and the merged imageof green fluorescent protein (GFP) and NSE (green and red) (FIGS. 11C,11D, and 11E, respectively). Magnification 100× for FIG. 11A; 630× forFIG. 11B; and 200× for FIGS. 11C, 11D, and 11E.

FIG. 12 is executed in color and depicts neural progenitor cells,stained for LacZ, seen in the tumor outgrowth migrating out from themain tumor mass at 10× (FIG. 12A) and 40× (FIG. 12B) magnification. Thesections were counterstained with hematoxylin.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the present invention are based on adult bone marrow as aviable alternative source of neural progenitor cells that may be used intherapeutic strategies for a variety of neuropathological conditions.

Any population of cells where neural progenitor cells are suspected ofbeing found may be used in accordance with the method of the presentinvention. Such populations of cells may include, by way of example,mammalian bone marrow, brain tissue, or any suitable fetal tissue.Preferably, cells are obtained from the bone marrow of a non-fetalmammal, and most preferably from a human. U.S. Pat. Nos. 6,204,053 B1and 5,824,489, the disclosures of which are hereby incorporated byreference, identify additional sources of cells that contain or arethought to contain stem cells; any of these cells may be used inaccordance with the methods of the present invention.

In one embodiment of the present invention, a mass of cells may beharvested or otherwise obtained from an appropriate source, such as, byway of example, adult human bone marrow. The mass of cells maythereafter be grown in a culture, and may be further subcultured wheredesirable, to generate further masses of cells. Any appropriate culturemedium may be used in accordance with the methods of the presentinvention, such as, by way of example, serum-free Dulbecco's modifiedEagle medium (DMEM)/F-12 medium.

The medium of the present invention may include various mediumsupplements, growth factors, antibiotics, and additional compounds.Supplements may illustratively include B27 supplement and/or N2supplement (both available from Invitrogen Corporation); growth factorsmay illustratively include fibroblast growth factor-2 (FGF-2), epidermalgrowth factor (EGF), and/or leukemia inhibitory factor (LIF); andantibiotics may illustratively include penicillin and/or streptomycin.In preferred embodiments of the present invention, growth factors areincluded in an amount of from about 15 ng/ml to about 25 ng/ml.Additional compounds suitable for use in the present invention mayinclude, but are in no way limited to, interleukin-3 (IL-3), stem cellfactor-1 (SCF-1), sonic hedgehog (Shh), and fms-like tyrosine kinase-3(Flt3) ligand. While not wishing to be bound by any theory, it isbelieved that these particular compounds may enhance the production ofspheres in accordance with the methods of the present invention.Additional or substituted supplements, growth factors, antibiotics, andadditional compounds suitable for use with the methods of the presentinvention may be readily recognized by one of skill in the art, andthese are contemplated as being within the scope of the presentinvention. In a most preferred embodiment of the present invention, aculture medium is DMEM/F-12 medium supplemented with B27, andadditionally includes 10 ng/ml of both FGF-2 and EGF, as well aspenicillin and streptomycin.

After a sufficient time period (generally from about three to about sixdays), clusters of neural progenitor cells (e.g., spheres) may form in aculture medium in which stem cells obtained as described above areincluded. Individual clusters of neural progenitor cells may be removedfrom the medium and sub-cultured separate from one another. Suchseparation may be repeated any desirable number of times to generate aclinically significant volume of neural progenitor cells. These neuralprogenitor cells may be capable of differentiating into a variety ofneural cells, such as, astrocytes, neurons, and oligodendroglia.

As used herein, a “clinically significant volume” is an amount of cellssufficient to utilize in a therapeutic treatment of a disease condition,including a neuropathological condition. Furthermore, as used herein,“treatment” includes, but is not limited to, ameliorating a disease,lessening the severity of its complications, preventing it frommanifesting, preventing it from recurring, merely preventing it fromworsening, mitigating an undesirable biologic response (e.g.,inflammation) included therein, or a therapeutic effort to effect any ofthe aforementioned, even if such therapeutic effort is ultimatelyunsuccessful.

The neural progenitor cells of the present invention possess a host ofpotential clinical and therapeutic applications, as well as applicationsin medical research. Two possible therapeutic mechanisms include: (1)using the cells as a delivery vehicle for gene products by takingadvantage of their ability to migrate after transplantation, and (2)using the cells to replace damaged or absent neural tissue, therebyrestoring or enhancing tissue function.

As discussed in the ensuing Examples, and with reference to the firsttherapeutic mechanism indicated above, the neural progenitor cells ofthe present invention are capable of “tracking” diseased or damagedtissue in vivo. The cells may therefore be used to aid in the targeteddelivery of various compounds useful in the treatment of diseased ordamaged tissue. Delivery of such compounds may be accomplished bytransfecting the cells with a gene that induces the cell to, forexample, constitutively secrete that compound itself, or promote abiochemical pathway that effects a local production of that compound.

Thus, in one embodiment of the present invention, neural progenitorcells may be transfected with or otherwise caused to carry a particulargene, as per any conventional methodology. Such methodologies mayinclude introducing a particular gene into the neural progenitor cellsas a plasmid, or, more preferably, using somatic cell gene transfer totransfect the cells utilizing viral vectors containing appropriate genesequences. Suitable viral vectors may include, but are in no way limitedto, expression vectors based on recombinant adenoviruses,adeno-associated viruses, retroviruses or lentiviruses, althoughnon-viral vectors may alternatively be used. In a preferred embodimentof the present invention, one employs adenovirus serotype 5(“Ad5”)-based vectors (available from Quantum Biotechnology, Inc.,Montreal, Quebec, Canada) to deliver and express desirable genesequences in the neural progenitor cells of the present invention. Oncecaused to carry the desired gene, the neural progenitor cells may beimplanted in or otherwise administered to a mammal.

By employing this therapeutic mechanism, the neural progenitor cells ofthe present invention may be used to treat a variety of pathologicalconditions; potentially any condition where mammalian neural tissue isdiseased or damaged to the point that neural progenitor cells will trackthe same. In the area of neuropathological disorders, this therapeuticmodality may be used in the treatment of numerous conditions, someexamples of which may include: brain tumors (e.g., by targeting thedelivery of cytokines or other agents that enhance the immune response,or by targeting the delivery of compounds that are otherwise toxic totumor cells); brain ischemia (e.g., by targeting the delivery ofneuroprotective substances such as brain-derived neurotrophic factor(BDNF), nerve growth factor (NGF), and neurotrophin-3, -4, and -5 (NT-3,NT-4, NT-5)); spinal cord injury (e.g., again, by targeting the deliveryof neuroprotective substances, or by targeting the delivery ofsubstances inducing neurite growth such as basic fibroblast growthfactor (bFGF), insulin-like growth factor-1 (IGF-1), and glial-derivedneurotrophic factor (GDNF)); and neurodegenerative disorders, such asAlzheimer's or Parkinson's Disease (e.g., again, by targeting thedelivery of neuroprotective substances or growth factors, or bytargeting the delivery of other neuroprotective factors such as amyloidprecursor proteins or protease nexin-1).

As discussed in the ensuing Examples, and with reference to the secondtherapeutic mechanism indicated above, the neural progenitor cells ofthe present invention are also able to replace neurons and glia in vivo.The cells may therefore be used to replace diseased or damaged neuraltissue, and, owing to the cells' additional capacity to track diseasedor damaged tissue in vivo, once administered, the cells may configurethemselves to an appropriate physiological site to effect thistherapeutic mechanism.

Given the ability of the neural progenitor cells of the presentinvention to replace lost or damaged neural tissue function, these cellsmay be useful in the treatment of numerous neuropathological conditions,many of which are similar to those enumerated above. By way of example,even in a state where the cells have not been transfected or otherwisecaused to carry a particular gene, the cells may be used in thetreatment of brain tumors, brain ischemia, spinal cord injury, andvarious neurodegenerative disorders.

Neural progenitor cells that are, in fact, transfected or otherwisecaused to carry a desirable gene may also provide the additional neuralcell function replacement capacity discussed in this mechanism; therebyimparting a dual treatment effect to the recipient. The dual treatmenteffect may include the replacement of lost or damaged cell function(e.g., as per the second therapeutic mechanism) in conjunction with thetargeted delivery of a beneficial compound to that same region (e.g., asper the first therapeutic mechanism). Therefore, in the illustrativeinstance of brain tumor treatment, the neural progenitor cells may betransfected with a gene that induces the secretion of cytokines (e.g.,tumor necrosis factor (TNF) or interleukin-1 (IL-1)), and implanted orotherwise administered to the brain of a recipient. Once administered,the cells may track the tissue damaged by the tumor, replacing at leasta portion of the lost brain function, while simultaneously secretingcytokines that may induce an immune response against the tumor cells.This dual treatment effect is further described in the ensuing Examples.

Neural progenitor cells developed through culture as described above maybe implanted in or otherwise administered to a mammal to effect thetherapeutic mechanisms previously discussed. Once implanted or otherwiseadministered, these cells may relocate to an area of diseased tissue,such as, but not limited to, brain tumors, tissue damaged by stroke orother neurodegenerative disease, and the like. Moreover, the neuralprogenitor cells may multiply in vivo, and may further follow diseasedtissue as it spreads (e.g., as a tumor spreads). Implantation may beperformed by any suitable method as will be readily ascertained withoutundue experimentation by one of ordinary skill in the art, such asinjection, inoculation, infusion, direct surgical delivery, or anycombination thereof.

EXAMPLES

All references cited herein are hereby incorporated by reference intheir entirety. The following examples are typical of the proceduresthat may be used to culture neural progenitor cells according to amethod of the present invention. Further examples are typical of theprocedures that may be used to perform gene transfer into these cellsand/or implant these cells into a patient to treat a neurologicaldisorder in accordance with another embodiment of the present invention.Modifications of these examples will be apparent to those skilled in theart.

Example 1 Isolation and Preparation of Neural Progenitor Cells

Whole bone marrow was harvested from the femurs of adult Fisher ratsbetween 16 and 24 weeks of age. Cultures were plated on poly-D-lysinecoated 24 well plates at a density of 106 cells per well. The cells werecultured in serum-free Dulbecco's modified Eagle medium (DMEM)/F-12medium supplemented with B27 (obtained from Gibco BRL; Gaithersburg,Md.), 20 ng/ml FGF-2 and 20 ng/ml EGF (both available from SigmaChemical Co.; St. Louis, Mo.; hereinafter “Sigma”), along withpenicillin and streptomycin (both available from Omega Scientific, Inc.;Tarzana, Calif.).

After four days in culture, numerous floating spheres of between about10 to about 100 cells were distinctly visible separate from anunderlying adherent monolayer (FIG. 1A). These spheres were collectedand sub-cultured separately (FIG. 1B). The cellular aggregates continuedto expand and the rate of proliferation remained stable even aftermultiple disassociations and passages. Numerous cells in these spherestested positive for nestin (FIGS. 2C and 2D), a known marker for neuralstem cells. U. Lendahl et al., Cell 60:585–595 (1990).

Spheres taken after four days of sub-culture were plated ontolaminin-coated 24 well plates in media devoid of growth factors. Thespheres attached and cells at the outer margins of each sphere began todevelop processes and migrate away from the primary site of attachment(FIG. 1C). Formed spheres detached from the bottom of the plates andthereafter remained free-floating (FIG. 1D); displaying multiplemorphologies (FIG. 4A). Neural progenitor cells expressed GFAP (FIGS. 4Band 4C) and the early neuronal marker NeuN (FIGS. 4D and 4E). Scatteredcells also expressed the later neuronal marker MAP2 (FIG. 4F). Bonemarrow-derived spheres were transplanted into the hippocampus of asyngeneic animal, and cells expressing NeuN were found (FIG. 4G); someof these cells intagrating into the hippocampal structure (FIG. 4H).Data was also collected utilizing alternate antibodies forimmunocytochemistry, including CNPase (FIG. 4I) and NF200 (FIGS. 4J and4K).

Neurospheres (i.e., cells derived from neural tissue) and bonemarrow-derived spheres were morphologically indistinguishable (FIGS. 2Aand 2B). The pattern of nestin expression was similar in both (FIGS. 2Cand 2D); although bone marrow-derived spheres expressed the ectodermalmarker vimentin (FIG. 3A) and also displayed a weak staining forfibronectin (FIG. 3B). Furthermore, the bone marrow-derived spheresexhibited strong expression of CD90 (FIG. 3C), and the majority of cellsin spheres also displayed nuclear expression of Neurogenin 1 (FIG. 3D).

Example 2 Gene Transfer into Neural Progenitor Cells UtilizingReplication-Deficient Adenoviral Vectors

Type 5 replication-deficient adenoviral vectors bearing either thereporter gene for β-galactosidase or the gene for the cytokine IL-12were used to infect neural progenitor cells in vitro. 24 hours followinginfection, successful gene transfer was confirmed using X-gal staining(X-gal Staining Assay Kit available from Gene Therapy Systems, Inc.; SanDiego, Calif.) for β-galactosidase-bearing adenoviral-infectedprogenitor cells, and an IL-12 Enzyme Linked Immunosorbent Assay(“ELISA” kit available from BD Pharmingen; San Diego, Calif.) for IL-12gene-bearing adenovirus-infected progenitor cells.

Successful gene transfer of β-galactosidase was confirmed by positivestaining for the X-gal and β-galactosidase-generated blue precipitate inthe β-galactosidase-bearing adenovirus-infected progenitor cells (FIG.5). Successful gene transfer of IL-12 was confirmed by the positivephotochromic ELISA reaction in media harvested from the IL-12gene-bearing adenovirus-infected progenitor cells (Table 1).

TABLE 1 Detection of IL-12 Secretion by ELISA IL-12 Detection by ELISACells infected in vitro with AdIL-12 >> 2 ng/ml Cells infected in vitrowith AdLacZ     4 pg/ml Mock infected cells Not detected

Example 3 Gene Transfer into Neural Progenitor Cells Utilizing aDouble-Mutated Herpes Simplex Virus Type I

A herpes simplex type I virus deleted for the genes encoding the latencyactivated transcript (LAT) and gamma 34.5 genes (virus denoted DM33) wasutilized. The virus contained the gene for GFP under the control of thepowerful LAT promoter, and was therefore able to confer constitutiveexpression of GFP into any successfully infected cell. This vector wasused to infect neural progenitor cells in vitro. 72 hours afterinfection, successful gene transfer was confirmed by viewing GFPexpression under a fluorescent light microscope (FIG. 6).

GFP expression was visible in neural progenitor cells 72 hours followinginfection with DM33. This confirmed the ability to successfully utilizeherpes simplex type I for gene transfer to neural progenitor cells.

Example 4 Neural Progenitor Cells Are Capable of Differentiating intoAstrocytes, Neurons, and Oligodendroglia

Neural progenitor cells were replated in vitro in culture media devoidof essential growth factors and supplemented with retinoic acid (a knownstimulator of differentiation). Culture surfaces were coated withpoly-D-lysine (available from Sigma) to promote attachment ofdifferentiating cells.

After three to four days, neural progenitor cells had attached to theculture surface and differentiated into astrocytes, neurons, andoligodendroglia. The presence of these cells was specifically confirmedby positive immunocytochemical staining populations for known markers ofall three lineages. Specifically, astrocytes in the culture populationwere positive for GFAP; neurons were positive for β-III tubulin; andoligodendroglia were positive for CNPase. This confirms the multipotencyand true progenitor nature of the neural progenitor cells of the presentinvention.

Example 5 Neural Progenitor Cells Track Spreading Brain Tumor Cells inVivo

Neural progenitor cells were infected with replication-deficientadenovirus bearing the gene for β-galactosidase as described in Example2 above. These cells were then transplanted intratumorally into C57bI/6mice bearing established GL26 brain tumors in their right cerebralhemispheres, respectively. After eleven days, the mice were euthanized,and their brains were immediately harvested, frozen, and sectioned usinga cryostat (available from Janis Research Company, Inc.; Wilmington,Mass.). The frozen sections were then stained using an X-gal stainingsolution to detect the presence of β-galactosidase-expressing neuralprogenitor cells within the brain tumors.

Neural progenitor cells were clearly visible within the main tumor mass.In addition, neural progenitor cells could clearly be seen trackingpockets of tumor cells that were migrating away from the main tumormass. This clearly demonstrated the ability of neural progenitor cellsto actively follow pockets of tumor cells that disseminate through thebrain.

Example 6 Neural Progenitor Cells Track Ischemic Brain Injury in Vivo

The middle cerebral artery of Wistar rats was occluded with a threadembolus for two hours. A drop in perfusion pressure verifiedeffectiveness of occlusion. Neural progenitor cells were infected withreplication-deficient adenovirus bearing the gene for β-galactosidase asdescribed in Example 2 above. The cells were infused intracraniallyeither immediately or two hours following middle cerebral arteryocclusion. After 48 hours, the rats were euthanized, and their brainswere immediately harvested, frozen, and sectioned. The fresh frozensections were then stained using an X-gal staining solution to detectthe presence of β-galactosidase-expressing neural progenitor cells inthe brain.

Neural progenitor cells were clearly identifiable in the sectionedbrains indicating that these cells can readily cross the blood brainbarrier. The transplanted cells were distributed throughout the ischemicpart of the brain, mostly as single cells infiltrating the pathologicaltissue (FIG. 8). While not wishing to be bound by any theory, it isbelieved that this may be part of the cells' response to chemotacticstimuli originating from the damaged tissue. The cells could also befound in normal parts of the brain and some cells were located in themeninges.

Example 7 Neural Progenitor Cells May Be Generated from Fetal BrainTissue

Neurospheres were generated from primary fetal brain culture, in amanner similar to that described with respect to bone marrow in Example1, above (i.e., cells were cultured in serum-free DMEM/F-12 mediumsupplemented with B27, 20 ng/ml FGF-2 and 20 ng/ml EGF, along withpenicillin and streptomycin). Neural stem cells grew into sphericalaggregates 2–3 days following harvest and cultured in growthfactor-supplemented media (FIG. 7A). These neurospheres were comprisedof neural progenitor cells expressing nestin (FIG. 7B).

The neural stem cells were re-plated in modified culture conditions,after the cells were induced to differentiate. Neurons expressed β-IIItubulin (FIG. 7C), astrocytes expressed GFAP (FIG. 7D), andoligodendrocytes expressed CNPase (FIG. 7E). Neural stem cells infectedin vitro with AdLacZ expressed β-galactosidase (FIG. 7F).

Example 8 Neural Progenitor Cells Track Tumor Cells in Vivo

Tumors from glioma-bearing mice inoculated with neural progenitorcell-LacZ were stained with X-gal and counter-stained with Neutral red.Four distinct patterns of tumor spread were detected and neuralprogenitor cells were found tracking migrating glioma in each case: (1)a thin outgrowth of tumor cells deep into adjacent normal brain; (2) adirect extension of tumor mass into adjacent tissue; (3) a migration ofglioma cells away from the primary tumor bed along a white matter tract;and (4) a tumor microsatellite independent of a main tumor mass (FIGS.9A–9D, respectively). Interspersed with the tumor cells depicted in thetumor microsatellite (FIG. 9D) were β-galactosidase positive neuralprogenitor cells; revealed with a high power photomicrograph (FIG. 9E).

Neural progenitor cells were inoculated into a cerebral hemispherecontralateral to an existing tumor. The progenitor cells were introducedinto the left cerebral hemisphere (FIG. 9F, left panel), butdemonstrated specific, non-random migration into the vicinity of thetumor in the opposite hemisphere (FIG. 9F, right panel and inset box).Neural progenitor cells appear blue (indicating expression ofβ-galactosidase), whereas tumor cells appear red (hypercellular areaswere stained intensively with neural red). Thus, neural progenitor cellsdisplay strong tropism for disseminating glioma in vivo.

Example 9 Neural Progenitor Cells Transfected with Cytokines InduceLocalized Immune Response in Vivo

Neural progenitor cells were transfected with genes that induced them tosecrete either IL-12, 3T3-IL-12 or LacZ, as described in Example 2,above. These neural progenitor cells were inoculated into theglioma-bearing brains of rats.

A flow cytometry analysis indicated robust intratumoral T-cellinfiltration in brains inoculated with neural progenitor cells secretingIL-12 and 3T3-IL-12 (FIG. 10A, left and center panels, respectively).However, intratumoral T-cell content of brains inoculated with neuralprogenitor cells secreting LacZ was much lower (FIG. 10A, right panel)and was comparable to infiltration seen in mock-transfected neuralprogenitor cells and saline-inoculated gliomas (data not shown).

Tumors treated with neural progenitor cells secreting IL-12 demonstratedrobust CD4+ and CD8+ T-cell infiltration (FIG. 10D, left and rightpanels, respectively), with numerous aggregates along the tumor/normaltissue boundary (FIG. 10D). Tumors treated with neural progenitor cellssecreting 3T3-IL-12 also demonstrated CD4+ and CD8+ T-cell infiltration(FIG. 10B, left and right panels, respectively), with positive cellsinterspersed in tumor tissue. However, tumors treated with neuralprogenitor cells secreting LacZ displayed negligible infiltration oftumors by CD4+ or CD8+ T-cells (FIG. 10C, left and right panels,respectively).

In a comparative analysis of T-cell infiltration in comparableoutgrowths from a primary tumor bed, tumor microsatellites in brainstreated with neural progenitor cells secreting IL-12 demonstrated robustT-cell infiltration, whereas those in brains treated with neuralprogenitor cells secreting 3T3-IL-12 did not (FIG. 10E, left and rightpanels, respectively).

Example 10 Neural Progenitor Cells Transfected with β-GalactosidaseTrack Tumor Cells in Vivo

RG2 tumor cells (100,000 in 5 ul of media) were stereotacticallyimplanted into the striatum of Wistar rats. Two days following tumorimplantation, 30,000 bone marrow derived cells infected with adenoviruscarrying the β-galactosidase gene were implanted into the same site. Theimmunohistological analysis was done 60 days following cellimplantation. As depicted in FIG. 12, the cells, stained for LacZ, canbe seen in the tumor outgrowth migrating out from the main tumor mass at10× (FIG. 12A) and 40× (FIG. 12B) magnification. The sections werecounterstained with hematoxylin.

While the description above refers to particular embodiments of thepresent invention, it will be understood that many modifications may bemade without departing from the spirit thereof. The accompanying claimsare intended to cover such modifications as would fall within the truescope and spirit of the present invention. The presently disclosedembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims, rather than the foregoing description,and all changes that come within the meaning and range of equivalency ofthe claims are therefore intended to be embraced therein.

1. A method to generate neural progenitor cells, the method comprising:culturing whole bone marrow from a mammal in a medium comprisingfibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) toproduce a neural progenitor cells.
 2. The method of claim 1, wherein thewhole bone marrow is obtained from an adult mammal.
 3. The method ofclaim 1, wherein the whole bone marrow is obtained from a fetus.
 4. Themethod of claim 1, wherein the medium is Dulbecco's modified Eaglemedium (DMEM).
 5. The method of claim 1, wherein the medium furthercomprises a supplement.
 6. The method of claim 5, wherein the supplementis selected from the group consisting of B27, N2, and combinationsthereof.
 7. The method of claim 1, wherein the medium further comprisesan additional compound selected from the group consisting ofinterleukin-3 (IL-3), stem cell factor-1 (SCF-1), sonic hedgehog (Shh),fms-like tyrosine kinase-3 (Flt3) ligand, leukemia inhibitory factor(LIF), and combinations thereof.
 8. The method of claim 1, wherein themedium further comprises an antibiotic.
 9. The method of claim 8,wherein the antibiotic is selected from the group consisting ofpenicillin, streptomycin, and combinations thereof.
 10. The method ofclaim 1, wherein clusters of the neural progenitor cells develop in themedium, and the method further comprises separating at least one clusterfrom the medium.
 11. The method of claim 10, further includingsubculturing the at least one cluster.
 12. The method of claim 11,wherein subculturing the at least one cluster further comprisesculturing the at least one cluster in a medium comprising FGF-2 and EGF.13. A method to generate neural progenitor cells, the method comprising:culturing whole bone marrow from a mammal in Dulbecco's modified EagleMedium (DMEM) comprising B27, penicillin, streptomycin, fibroblastgrowth factor-2 (FGF-2) and epidermal growth factor (EGF), whereinsphere clusters of the neural progenitor cells develop in the medium,and the method further comprises separating at least one sphere clusterfrom the medium and subculturing the at least one sphere cluster in amedium comprising FGF-2 and EGF to produce neural progenitor cells.